ADP-ribosylation factors (ARFs) are multi-functional, multi-domain, proteins that regulate vesicular trafficking in the ER and Golgi (and elsewhere). ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase-activating proteins (GAPs). Two general types of GEPs have been recognized, a family of ~200-kDa proteins that are inhibited by BFA (a drug that interferes with protein secretion and causes reversible disintegration of Golgi cisternae) and smaller ~55-kDa GEPs that are BFA-resistant. All have so-called Sec7 domains of ~200 amino acids that are responsible for GEP activity, as well as its BFA inhibition. To assess the effects of structure outside the Sec7 domain, we had compared the activities of cytohesin-1, a ~55-kDa GTP, and its Sec7 domain (C-1Sec7) toward several ARF and ARL proteins. ARL1, first described by other workers in 1991 as an ARF-related protein that did not activate cholera toxin, is 56% identical in amino acid sequence to ARF1. Both cytohesin-1 and C-1Sec7 activate ARF1, but only the latter activates ARL1, i.e., substrate specificity is influenced by structure outside of the catalytic Sec7 domain. Chimeric proteins composed of sequences from ARF1 and ARL1 were prepared several years ago to show that different regions of ARF1 are responsible for the activation of cholera toxin and phospholipase D and to define regions criticals for the interaction with arfaptin. We used those and additional constructs in recent studies to identify multiple structural elements in ARF1 that participate in functional interactions with cytohesin-1. For example, the ARF N-terminus, which was required for cytohesin-1 action, was not necessary for that of C-1Sec7. The data from these experiments mean that the model based on a crystal structure of the ARNO Sec7 domain (which is 87% identical to C-1Sec7) associated with an ARF1 lacking the first 17 residues does not reflect completely, or perhaps, correctly, interactions in the ARF-cytohesin complex. - GTP-binding proteins, ARF, ARL, phospholipase D, cholera toxin